The present invention concerns a new branched natural soluble polysaccharide comprising a main chain having repeating side chains which are only made of lactose units, possibly substituted. The present invention also concerns the microorganism by which this branched polysaccharide may be obtained and...http://www.google.com/patents/US5955602?utm_source=gb-gplus-sharePatent US5955602 - Branched lactose containing polysacharides and compositions containing them

The present invention concerns a new branched natural soluble polysaccharide comprising a main chain having repeating side chains which are only made of lactose units, possibly substituted.

The present invention also concerns the microorganism by which this branched polysaccharide may be obtained and the food composition, the cosmetical composition and the pharmaceutical composition comprising said branched polysaccharide and/or microorganism.

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Claims(11)

We claim:

1. A purified branched natural soluble polysaccharide consisting of a main chain having two or more side chains, wherein said side chains consist only of lactose units or units of the formula ##STR7## wherein Gal=galactose, Glc=glucose, R1 =hydrogen or N-acetylgalactosamine, R2 =hydrogen, N-acetylneuraminic acid or HSO3, R3 =hydrogen or fucose.

2. The branched polysaccharide according to claim 1, which has the structure of the following formula: ##STR8## wherein n>1, Gal=galactose, Glc=glucose, R1 =hydrogen or N-acetylgalactosamine, R2 =hydrogen, N-acetylneuraminic acid or HSO3, R3 =hydrogen or fucose, subscript "f" denotes a furanose form.

3. The polysaccharide according to claim 2, wherein each of R1, R2, and R3 is hydrogen, and which polysaccharide comprises glucose and galactose in the molecular ratio 1:1.1.

4. A food composition comprising: a purified branched polysaccharide consisting of a main chain having two or more side chains, wherein said side chains consist only of lactose units or units of the formula ##STR9## wherein Gal=galactose, Glc=glucose, R1 =hydrogen or N-acetylgalactosamine, R2 =hydrogen, N-acetylneuraminic acid or HSO3, R3 =hydrogen or fucose; and an appropriate edible carrier.

5. The food composition according to claim 4, wherein said branched polysaccharide has the structure of the following formula: ##STR10## wherein n>1, Gal=galactose, Glc=glucose, R1 =hydrogen or N-acetylgalactosamine, R2 =hydrogen, N-acetylneuraminic acid or HSO3, R3 =hydrogen or fucose, subscript "f" denotes a furanose form; and an appropriate edible carrier.

6. A cosmetic composition comprising: a purified branched polysaccharide consisting of a main chain having two or more side chains, wherein said side chains consist only of lactose units or units of the formula ##STR11## wherein Gal=galactose, Glc=glucose, R1 =hydrogen or N-acetylgalactosamine R2 =hydrogen, N-acetyineuraminic acid or HSO3, R3 =hydrogen or fucose; and a cosmetically acceptable carrier.

7. The cosmetic composition according to claim 6, wherein said branched polysaccharide having the structure of the following formula: ##STR12## wherein n>1, Gal=galactose, Glc=glucose, R1 =hydrogen or N-acetylgalactosamine, R2 =hydrogen, N-acetylneuraminic acid or HSO3, R3 =hydrogen or fucose, subscript "f" denotes a furanose form; and a cosmetically acceptable carrier.

8. The cosmetic composition according to claim 6, wherein said composition is selected from the group consisting of mouth rinse, toothpaste, tooth gel, chewing gum, cream, ointment, and balsam.

9. A pharmaceutical composition comprising: a purified branched polysaccharide consisting of a main chain having two or more side chains, wherein said side chains consist only of lactose units or units of the formula ##STR13## wherein Gal=palactose, Glc=glucose, R1 =hydrogen or N-acetylgalactosamine, R2 =hydrogen, N-acetylneuraminic acid or HSO3, R3 =hydrogen or fucose; and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, wherein said composition is selected from the group consisting of a capsule, syrup, powder, and a tablet.

11. The pharmaceutical composition according to claim 9, wherein said branched polysaccharide having the structure of the following formula: ##STR14## wherein n>1, Gal=galactose, Glc=glucose, R1 =hydrogen or N-acetylgalactosamine, R2 =hydrogen, N-acetyineuraminic acid or HSO3, R3 =hydrogen or fucose, subscript "f" denotes a furanose form; and a pharmaceutically acceptable carrier.

Description

FIELD OF THE INVENTION

The present invention concerns a new branched polysaccharide, a microorganism producing it, the food composition, the pharmaceutical composition and the cosmetical composition containing them.

BACKGROUND OF THE INVENTION

Biological communication (the possibility for a cell to recognize a molecule or another cell) is a central phenomenon in pathological as well as in normal states.

Among the various mechanisms of molecular recognition between cells, and/or between cells and molecules, the binding of specific glycosidic structures by specialized proteins (lectins) is today considered as a major molecular recognition system.

The lectins may be bound specifically and non-covalently to well-defined glycosidic sequences.

Some lectins are bound, for example, to oligosaccharides which contain elevated mannose amounts, to structures carrying sialic acids, or to fucosylated glycosides.

Between these bacterial cells, the interaction is often obtained by a non-covalent bond between a β-galactoside lectin on one cell and a glycosidic receptor on another cell (ref. 1).

Most infectious diseases are initiated by the adhesion of pathogenics agents (such as Actinomyces naeslundii, Fusobacterium nucleatum, Bacteriodes intermedius, Salmonella typhimurium, Vibrio Cholera, Campylobacter jejuni, Bacteriodes, Fusobacteria, Clostridia, Shigella, Yersinia, and Helicobacter pylori, etc.) to the epithelial cells of the mucosa of its host, which allows then the colonisation of the animal tissues.

This adhesion is often obtained by a binding between a β-galactoside lectin located at the surface of this pathogeneous agent and a glycosidic receptor located at the surface of the epithelial cell (ref. 2).

Various cells of the immune system (lymphocytes T and B. macrophages, neutrophils) are known either to be able to bind β-galactoside lectins or to express at their surface such lectins of the galectin family.

In addition, some epithelial cells such as intestinal cells or keratinocytes produce these galectins which can also coat Langerhans cells, and immunoglobulins such as IgE can specifically bind to galectins (ref. 3, 4 and 5).

STATE OF THE ART

There have been many prior studies upon polysaccharides produced by micro-organisms and, in recent years, there have been several reports of studies on the structure of exocellular polysaccharides obtained from lactic acid bacteria and on their biological activities.

A polysaccharide consisting of galactose, glucose and N-acetylgalactosamine (2:1:1) is obtained by the strains of Streptococcus thermophilus CNCM I-733, CNCM I-734 and CNCM I-735 (ref. 6 and 7);

a polysaccharide consisting of galactose only is obtained by the strain Lactococcus cremoris H414 (ref. 8);

a polysaccharide consisting of galactose, glucose and rhamnose (5:1:1) is obtained by the strain Lactobacillus bulgaricus rr (ref. 10);

a polysaccharide consisting of glucose, rhamnose, 1-phosphoglycerol and a O-acetyl group (3:2:1:0.85) is obtained by the strain Lactobacillus sake 0-1 (ref. 11).

On the other hand, other polysaccharides obtained by a few strains of Lactobacillus helveticus were studied, but their structural characterization were never performed. For example, a polysaccharide of unknown structure consisting of glucose and galactose (2:1) used as an anti-tumor agent is obtained by the strain Lactobacillus helveticus var. jugurti No 851 "FERM BP-66 (FERM-P No 5851)" (ref. 12 and 13). Similarly, a polysaccharide of unknown structure consisting of galactose, glucose and N-acetylgluccsamine (2.5-3.5:2.5-3.5:1) used in treating inflammation and to accelerate bone marrow growth is obtained by the strain Lactobacillus helveticus MIKI-010 (ref. 14).

AIMS OF THE INVENTION

The present invention aims to provide a new branched polysacharide and/or the microorganism producing it, which can be used to inhibit the binding between β-galactoside lectins and their receptor(s).

Another aim of the invention is to provide food compositions comprising said polysaccharide and/or microorganism, having improved organoleptic and texture properties.

A further aim of the invention is to provide a pharmaceutical composition and/or cosmetical composition, comprising said polysaccharide and/or microorganism.

A last aim of the invention is to provide a polysaccharide which can be used as an intermediate product for the production of polymerized derivatives of gangliotriose, Sd-a blood group, or sialyl- and sulfated-Lewis X.

DESCRIPTION OF THE INVENTION

The present invention concerns a new natural soluble branched polysaccharide comprising a main chain having repeating side chains which are only made of lactose units, possibly substituted.

According to a preferred embodiment of the present invention, the branched polysaccharide corresponds to the following formula: ##STR1## where n>1, Gal=galactose,

Glc=glucose,

R1 =Hydrogen or GalNAcβ1 (N-acetylgalactosamine)

R2 =Hydrogen, NeuNAcα2 (N-acetylneuraminic Acid) or HSO3

R3 =Hydrogen or Fucα1 (fucose)

When R1 =R2 =R3 =Hydrogen, the branched polysaccharide is characterized by the following physicochemical properties:

molecular weight higher than 2,000,000, soluble in water and solutions containing less than 20% trichloroacetic acid,

insoluble in alcohol and in acetone,

neutral property,

the freeze-dried product is in the form of white powder,

component sugars and compositional ratio:Glucose:Galactose (1:1.1).

When R1 is GalNAcβ1 (N-acetyl galactosamine) and R2 =R3 =Hydrogen, the branched polysaccharide is a derivative of the gangliotriose determinant which is advantageously obtained from an intermediate product (the polysaccharide with R1 =R2 =R3 =Hydrogen) by the methods described in documents 15 and 16.

When R1 is GalNAcβ1 (N-acetyl galactosamine), R2 is NeuNAcα2 (N-acetyl neuraminic Acid) and R3 is Hydrogen, the branched polysaccharide is a derivative of the blood group Sd-a determinant, which is advantageously obtained from an intermediate product (the polysaccharide with R1 =R2 =R3 =Hydrogen) by the methods described in documents 17 and 18.

When R1 is Hydrogen R2 is NeuNAcα2 (N-acetylneuraminic Acid) or HSO3 and R3 is Fucα1 (fucose), the branched polysaccharide is a derivative of the pharmaceutical products sialyl- or sulfated- Lewis X described in documents 19 and 20.

The bindings between Gal β 1-4 Glc and R1, R2 are advantageously obtained from an intermediate product (the polysaccharide with R1 =R2 =R3 =Hydrogen) by the method described in document 17.

The present invention also concerns the microorganism producing the branched polysaccharide having R1 =R2 =R3 =Hydrogen.

Advantageously, said microorganism corresponds to a strain of Lactobacillus helveticus, preferably the strain of Lactobacillus helveticus CNCM I-1449.

A deposit of this microorganism has been made according to the Budapest Treaty on Jul. 27, 1994 under acession number CNCM I-1449, at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX 15, FRANCE.

The present invention also concerns the food having improved organoleptic and texture properties comprising the polysaccharide and/or the microorganism according to the invention.

Preferably, said food composition is a set-style acidified milk or a stirred acidified milk.

Another aspect of the present invention concerns a cosmetic composition comprising the polysaccharide and/or the microorganism according to the invention.

According to a preferred embodiment of the invention, said cosmetic composition is a cosmetic product intended for buccal hygiene, choosen among the group consisting of a tooth paste, tooth gel, mouth rinse, chewing-gum and/or tablet.

According to another preferred embodiment of the present invention, said cosmetic composition is a product intended for skin hygiene, choosen among the group consisting of a cream, ointment or balsam.

Another aspect of the present invention concerns a pharmaceutical composition comprising the polysaccharide and/or the microorganism according to the present invention.

Advantageously, said composition is a antidiaorrheic product choosen among the group consisting of a capsule, syrup, powder and/or tablet.

A last aspect of the present invention concerns a diagnostic and/or analytic device comprising the branched polysaccharide according to the invention for the trapping of specific molecules and/or microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the chromatographic (FPLC) analysis of the polysaccharide according to the invention.

FIG. 2 represents six major fractions separated by gel filtration from the polysaccharide hydrolysate.

The present invention concerns a new natural soluble branched polysaccharide with a main chain, having repeating side chains which are only lactose units, said lactose being possibly substituted.

The "branched polysaccharide" according to the invention is a saccharide having more than 10 repeating units, preferably more than 40 repeating units.

Said branched polysaccharide preferably has the structure of a "polymer" consisting of the repetition of identical single "units" comprising one side chain 2 branched on a main chain 1 as described below: ##STR2##

The production and the physico-chemical structure of a polysaccharide according to the invention will be described hereafter.

1. PREPARATION AND PURIFICATION OF THE EXOPOLYSACCHARIDE PRODUCED BY LACTOBACILLUS HELVETICUS CNCM I-1449

1.1 Fermentation Conditions.

Lactobacillus helveticus CNCM I-1449 was a ropy strain from the Nestle strain collection. Among the 168 strains of L. helveticus of the Nestle collection, only 2 strains produce exopolysaccharides.

The growth medium was 10% reconstituted skim milk heat-treated (115° C., 35 min) for sterilization prior to fermentation. A one-liter scale fermentor with a magnetic stirrer was used for regulating the pH during the fermentation. The pH was maintained at 5.5 by using 2N sodium hydroxide. Stirring rate was maintained at 60 RPM. Incubation was made at 40° C. The amount of starter culture inoculated to the medium was 1%. During the fermentation (time=6 h, 9 h and 24 h), several samples were taken and frozen for further analysis and polysaccharide extraction.

1.2. Extraction of the Polysaccharide.

An equal volume or trichlorcacetatic acid (40%) was added to the sample to remove proteins by precipitation, followed by centrifugation (17,000 g, 20 minutes). To the supernatant fraction containing polysaccharides, the same volume of acetone was added. Precipitated polysaccharides were then separated by centrifugation (17,000 g, 20 minutes).

The resulting precipitated fraction was dissolved in distilled water and pH was adjusted to 7.0 with sodium hydroxide solution. After dialysis against distilled water (overnight), insoluble substances were removed by ultracentrifugation (110,000 g, 1 hour).

The supernatant fraction containing polysaccharides was lyophilized and crude dehydrated polysaccharides were finally obtained. Total neutral sugar content was determined by the phenol-sulphuric acid method.

1.3. Size of the Exopolysaccharide.

Filtration was conducted to confirm purity and estimate the molecular weight of polysaccharides using FPLC system (Pharmacia). The column used was Superose 6 (1.0 cm×30 cm) (FIG. 1). 200 μl samples containing 200-400 μg dehydrated polysaccharides were applied on to the column, eluted with 50 mM phosphate buffer at pH 7.2 at the rate of 0.5 ml/min. Fractions (1.0 ml) were collected in tubes. Polysaccharide content in each tube was determined as total neutral sugar by the phenol-sulphuric acid method.

1.4. Monosaccharide Composition.

The monosaccharide composition of a freeze-dried polysaccharide was analyzed using gas-liquid chromatography technique (ref. 21).

The exopolysaccharide obtained from the spent culture medium was examined after extraction from three samples (time=6 h, 9 h and 24 h) . The yield was found to increase as a function of the fermentation time, but the size as well as the monosaccharide composition of the polymer were found invariable:

FIG. 1 shows the elution and the purity of the polysaccharide obtained at t=24 h, by FPLC analysis (with a column of Superose 6). The polysaccharide was eluated at around the exclusion limit (approximately 2×106 M.W.)

The polysaccharide described above was also produced during fermentations in set-style conditions, by Lactobacillus helveticus CNCM I-1449 alone, or by this strain used together with a strain of Streptococcus thermophilus (for example S. thermophilus YS4).

For this purpose, the growth medium was 10% reconstituted MSK (skim milk powder: 100 g/l and yeast extract: 1 g/l) heat-treated (115° C., 35 min) for sterilization prior to fermentation. The typical sample size was 250 ml, the incubation was made at 40° C. and the amount of starter culture inoculated to the medium was 1%. The yields of pure polysaccharide obtained in such conditions were the following:______________________________________Strains in the Fermentation Time Pure YieldStarter Culture (hours) (mg/l)______________________________________L. helveticus 8 85CNCM I-1449 16 140L. helveticus 4 23CNCM I-1449 & S. 8 81thermophilus YS4______________________________________

2. METHODS USED FOR THE STRUCTURAL CHARACTERIZATION OF THE EXOPOLYSACCHARIDE PRODUCED BY LACTOBACILLUS HELVETICUS CNCM I-1449.

2.1. Monosaccharide Analysis.

Polysaccharide (0.1 mg) was methanolysed (methanolic 0.5 M HCl, 24 h, 80° C.) and (one night at room temperature). The trimethylsilylated methyl glycosides were analysed by gas phase chromatography (Varian 3400) using a BP1 fused-silica capillary column (25 m×0.32 mm, SGE). The elution was performed by applying on the column a temperature gradient from 120° C. to 240° C. at 2° C. min1. The absolute configuration of the monosaccharides was determined by GLC of the trimethylsilylated (N-reacetylated) (-)-2-butyl glyclosides.

2.2 Methylation Analysis.

Samples (native polysaccharide and oligosaccharide-alditols) were permethylated, and methylated products were subjected either to methanolysis or acid hydrolysis (trifluoroacetic acid 4N, 4 h, 100° C.) followed by reduction with BD4 Na.

The partially methylated and acetylated (pyridine anhydride acetic 1:1) methyl glycosides and alditols were identified by GLC (BP1 column) and GLC MS in e.i. mode on a Nermag R10-10S mass spectrometer using an electron energy of 70 ev and an ionizing current of 0.2 mA.

The eluted fractions were immediately neutralized with M acetic and lyophilized. The fractions were successively desalted on a column (6×1 cm) of Dowex 50×8 (H') resin and on a column of Fractogel (55×2 cm) using deionised water as eluent.

All the spectra were obtained at a probe temperature of 353° K. One dimensional spectra were obtained with a spectral width of 3000 Hz for a 16K frequency-domain points and time-domain data points giving a final digital resolution of 0.365 Hz/point.

The 100 MHz 13 C-NMR experiments were obtained with the standard Brucker pulse program Powgate with 1 H composite pulse decoupling. The spectral width was 22.727 Hz for a 32K frequency-domain data points and time-domain data giving a final digital resolution of 1.387 Hz/point; a ninety-degree pulse (6 μs) and 1 s recycle delay were used. The chemical given relative to the signal of the methyl group of acetone (δ 2.225 for 1 H and δ 31.55 for 13 C).

The 2D-homonuclear COSY 45, COSY with simple, double-and triple relay transferts were performed by use of the standard Bruker pulse program library or the programs given by B. Perly (C.E.A., Saclay). For all RCT experiments, refocusing delays of 35 ms were chosen and the relaxation delay was 2 s. In all these experiments, the spectral width was 1840 Hz, the 1 H ninety-degree pulse was 10.6 μs; 256 W×2K data matrices were acquired, which were zero-filled prior to Fourier transform, to obtain a 1K×2K spectral data matrix and a sine-bell squared function was used in both dimensions.

The 2D-13 C/1 H COSY experiments were performed with simultaneous suppression of 1 H homonuclear couplings by use of the standard Bruker pulse program XHCORRD. Refocusing delays were adjusted to an average 1 jC.H coupling constant of 150 KHz. 1 H and 13 C ninety-degree pulse width were 10.6 and 6 μs. The relaxation delay was 0.8 s. 128 W×4K data matrix was acquired, which was zero-filled prior to Fourier transform, to obtain a 512 W×4K spectral data matrix. An exponential function (LB=1 Hz) for 13 C-subspectra and a sine-bell function for 1 H-spectra were applied to enhance the signal to noise ratio.

3. STRUCTURE OF THE EXOPOLYSACCHARIDE PRODUCED BY LACTOBACILLUS HELVETICUS CNCM I-1449.

3.1. Isolation and Composition Analysis of the Polysaccharide.

GLC. analysis of the trimethylsilylated glycosides and (-)-2-butyl glyosides has confirmed the presence of D-galactose and D-glucose in a molar ratio 1:1.

Examination of the relayed COSY spectra of oligosaccharide alditols IV B and II A obtained by partial acid hydrolysis (see below) leads to the obvious identification of the β-D-Galp. As demonstrated by the COSY spectrum of the polysaccharide, the H-2 and H-3 of the β-D-Galp F residue exhibit a strong coupling constant which does not allow an to analysis of their multiple patterns.

Correlation peaks observed in the 1 H-13 C heteronuclear COSY spectrum show that one of the proton resonances (5.158 ppm) is connected to the carbon resonance deshielded at 108.95 ppm that proves a β-anomeric configuration for the Galf A residue.

Most of the proton resonances may be assigned in the homonuclear COSY spectrum except for the H-5 and H-6 spin systems of the β-D-Galp F residue. The carbon resonances may also be assigned by direct correlation to their attached protons.

The two remaining unassigned carbons at 73.52 and 61.12 ppm were deduced to correspond with the C-5 and C-6 atom resonances of β-D-Galp F residue.

In summary, all these-assignments clearly furnish the substitution pattern of each sugar unit, according to their downfield shifted carbon resonances:

C-3 and C-5 for β-D-Galf A,

C-3 for α-D-Glcp C,

C-3 for α-D-Galp D,

C-3 for β-D-Glcp B and

C-4 for β-D-Glcp E.

The sixth sugar unit β-D-Galp F, which does not possess any downfield shifted 13 C resonance, occurs consequently in a non-reducing position.

In order to elucidate the position of the branched terminal galactosyl residue, oligosaccharides were produced by partial acid hydrolysis of the native polysaccharide. Six major fractions were separated by gel filtration on Fractogel HW40 F from the hydrolysate (FIG. 2).

Two of them (fractions II and IV) were subjected to HPAE-PAD chromatography (FIG. 3, 4 and table 3) and the structure of subfractions denoted II A, IV A and IV B was investigated both by NMR and methylation analysis.

Oligosaccharide-alditol IV A contains two Glc residues and one hexitol residue, as shown by the 1 H spin system depicted in the two-steps relayed COSY spectrum. The two Glc residues occur at the non-reducing terminal position, as confirmed by the methylation analysis which furnished 2, 3, 4, 6-tetra-O-methyl glucitol. The last derivative shows a pattern corresponding to a C-3 and C-5 substitution characteristic of a furanic sugar. From the previous n.m.r. data, this hexitol originates from the β-D-Galf A unit. These findings lead to the following structure: ##STR3##

It must be noted that due to the symmetry displayed by the 1 H spin system of galactitol, the 1 H NMR assignment of the compound was achieved after the elucidation of compound IV B structure.

The attachment of β-D-Glcp at C-3 of Gal-ol is not susceptible to modify dramatically the chemical shift of the H-1 and H-2 resonances of the hexitol, when compared with the NMR data from compound IV A. Therefore, the two signals at 3.77 and 4.056 ppm can be assigned to H-1 and H-2 of Gal-ol, whereas the H-5 and H-6 atoms resonances are upfield shifted at 4.149 and 3.68 ppm, by comparison with commpound IV A (Table 3).

In the NMR spectra of IV A and IV B, the H-5 resonance of Gal-ol is the most upfield shifted atom resonance of the molecule and this observation is in agreement with the assignments proposed by others.

Oligosaccharide-alditol II A (Table 2) contains 2 β-D-Glcp, 1 β-D-Galp and 1 α-D-Glcp residues, whereas the presence of C-3 and C-5 substituted Gal-ol can be easily deduced by the similarity in chemical shifts of H-1 to H-6 of Gal-ol in compound 1. Since the two β-D-Glcp units and the non-reducing terminal β-D-Galp have already been located in oligosaccharide-alditols IV A and IV B, and according to the fact that the β-D-Glcp B residue is C-3 substituted, the oligosaccharide-alditol II A possesses the following structure: ##STR5##

The sixth sugar unit, α-D-Galp, was not observed among the products of the partial hydrolysis. Nevertheless, the fact that the α-D-Glcp unit C is C-3 substituted and that the only β-D-Galp present in the polysaccharide occupies the non-reducing position (from the NMR data) lead us to conclude that the α-D-Galp is attached to this α-D-Glcp residue.

Therefore, the structural unit of the polysaccharide was defined as follows: ##STR6##

The compositions comprising the polysaccharide and/or microorganism according to the invention are described in the following non limiting examples.

EXAMPLE 1

Set-Style Acidified Milk.

Set-style acidifed milk comprising the L. helveticus strain according to the invention and two S. thermophilus strains, traditionnaly used for the production of a set-style yoghourt, was obtained by the following process.

A deposit of the microorganism has been made according to the Budapest Treaty on May 18, 1994, for the Streptococcus strains and on Jul. 27, 1994, for the Lactobacillus strain at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX 15, FRANCE.

The sterilized milk was inoculated with 1% of the third culture of each S. thermophilus strain and with 2% of the second culture of L. helveticus strain taken at the medium coagulation stage.

The milk was incubated at 42° C. and at a pH around 4.65, and then cooled at a temperature of 4° C.

EXAMPLE 2

Acidified Whey Milk.

Whey milk comprising the L. helveticus strain acording to the invention and two S. thermophilus strains, traditionnaly used for the production of a yoghourt, was obtained by the following process.

A sweet lactoserum powder was reconstituted at 12.5% in water.

40 liters of this whey was pasteurized at 92° C. for six minutes, homogeneized at 75° C. and 150 bars (two levels) and cooled at a temperature around 42° C.

A deposit of the microorganism has been made according to the Budapest Treaty on May 18, 1994, for the Streptococcus strains and on Jul. 27, 1994, for the Lactobacillus strain at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX 15, FRANCE.

The sterilized milk was inoculated with 1% of the third culture of each S. thermophilus strain and with 2% of the second culture of L. helveticus strain taken at the medium coagulation stage.

The whey milk was incubated at 42° C. and at a pH around 4.65, and then cooled at a temperature of 4° C.

EXAMPLE 3

Stirred Acidified Milk.

A stirred acidifed milk comprising the L. helveticus CNCM I-1449 strain according to the invention and two commercialized S. thermophilus strains, traditionnaly used for the production of a stirred yoghourt, was obtained by the following process.

The milk was obtained from a whole milk comprising 3.7% fats, by the addition of 2.5% skimmed milk powder.

40 liters of this milk was pasteurized at 105° C. for two minutes, homogeneized at 75° C. and 300 bars (first level) and cooled at a temperature around 43° C.

A deposit of the microorganism has been made according to the Budapest Treaty on May 18, 1994, for the Streptococcus strains and on Jul. 27, 1994, for the Lactobacillus strain at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 PARIS CEDEX 15, FRANCE.

The sterilized milk was inoculated with 1% of the third culture of each S. thermophilus strain and with 2% of the third culture of L. helveticus strain taken at the medium coagulation stage.

The milk was incubated at 43° C. and at a pH around 4.65, and then cooled at a temperature of 4° C. during stirring.

The following table 5 represents the properties of the obtained products.

A pharmaceutical composition was obtained as a capsule which was made with gelatine and water, and which contained from 5 to 50 mg of the exopolysaccharide according to the present invention. Alternatively, powdered tablet formulations can be obtained directly from the acidified cultured milks described in the above examples 1, 2 and 3, by freeze-drying these fermented milks and whey.